EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity Michael Peled, Matthew A. Dragovich, Kieran Adam, Marianne Strazza, Anna S. Tocheva, Irving E. Vega and This information is current as Adam Mor of September 26, 2021. J Immunol published online 1 October 2018 http://www.jimmunol.org/content/early/2018/09/28/jimmun ol.1800839 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/09/28/jimmunol.180083 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 1, 2018, doi:10.4049/jimmunol.1800839 The Journal of Immunology EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity Michael Peled,*,† Matthew A. Dragovich,* Kieran Adam,* Marianne Strazza,* Anna S. Tocheva,* Irving E. Vega,‡ and Adam Mor*,x Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell– mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important Downloaded from regulator of T cell cytotoxicity and further studies should evaluate its role in T cell–mediated inflammation. The Journal of Immunology, 2018, 201: 000–000. he immunological synapse (IS) is the interface between Most of the immune checkpoints are coinhibitory receptors that T cells and APC. The IS consists of segregated clusters of directly inhibit signaling downstream of the TCR. T proteins involved in T cell activation. A key member of Programmed cell death 1 (PD-1) is a typical coinhibitory re- http://www.jimmunol.org/ these clusters is the TCR that is engaged by an Ag-loaded MHC ceptor,expressedonactivatedTcellsandclustersintheIS on the APC. Productive interactions initiate signaling cascades alongside the TCR upon engagement by its ligands, PD-1 ligand 1 that result in T cell adhesion, proliferation, and cytokine secretion. (PDL1) or PDL2 (4). PDL1 is widely expressed on both hema- When the IS is composed of a cytotoxic T cell and a corresponding topoietic and nonhematopoietic cells, including tumor cells, target cell (such as a tumor or virally infected cell), lytic granules whereas PDL2 is mainly expressed on APC (5–9). Upon ligation, are released to eliminate the counter cells. To execute these func- PD-1 recruits the phosphatase SHP2 to dephosphorylate phos- tions, a stable synapse between the T cell and the target cell must phoproteins downstream of the TCR and to inhibit T cell acti- be formed. Indeed, TCR-induced synapses are characterized by vation (10–15). PD-1 knockout mice develop inflammatory reorientation of the microtubule-organizing center and substantial cardiomyopathy and glomerulonephritis (16, 17), and PD-1 block- by guest on September 26, 2021 actin accumulation at the boundaries of the contact region (1–3). ade induces potent antitumor immune response in patients with Whereas T cell functions are required for mounting protective various types of malignancies (18, 19). immune responses, it can also result in autoimmunity unless tightly We have recently used affinity purification mass spectrometry regulated. Accordingly, immune responses are restrained by a (MS) to discover proteins, besides SHP2, that interact with the plethora of mechanisms, including thymic clonal deletion of self- intracellular tail of PD-1 and regulate its function (14). Because reactive T cells, inhibitory cells such as regulatory T cells, and anti- recruitment of PD-1 to the IS is critical for its function (4), we inflammatory cytokines (i.e., IL-10 and TGF-b). Another important used this data set to screen for proteins that interact with PD-1 and mechanism to restrict immune responses is immune checkpoints. that also regulate IS formation through cytoskeleton organization. Multiple proteins were uncovered, including Swiprosin-1/EF hand domain family member D2 (EFHD2). EFHD2 knockdown (KD) *Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016; †Pulmonary Department, The Chaim Sheba Medical Center, Ramat Gan blocked PD-1 inhibitory functions in T cells through inhibition of 52620, Israel; ‡Department of Translational Science and Molecular Medicine, Col- stable IS formation. However, EFHD2 was also required for lege of Human Medicine, Michigan State University, Grand Rapids, MI 49503; and x T cell–mediated cytotoxicity and for antitumor immune response, Columbia Center for Translational Immunology, Columbia University Medical Center, New York, NY 10032 implying a pervasive role for this protein in IS formation. ORCID: 0000-0003-3106-1183 (M.S.). Received for publication June 18, 2018. Accepted for publication August 24, 2018. Materials and Methods This work was supported by a grant from the National Institutes of Health (R01 General reagents AI25640). RPMI 1640 medium, DMEM, Dulbecco’s PBS, and FBS were purchased Address correspondence and reprint requests to Dr. Adam Mor, 630 W 168 Street, from Life Technologies. Opti-MEM I was purchased from Invitrogen. PH8-406, Columbia University Medical Center, New York, NY 10032. E-mail Ficoll–Paque was purchased from GE Healthcare. Staphylococcus en- address: [email protected] terotoxin E (SEE) was acquired from Toxin Technology. BCA assay was The online version of this article contains supplemental material. purchased from Pierce Biotechnology. Poly-L-lysine, puromycin, and fibro- Abbreviations used in this article: EFHD2, EF hand domain family member D2; IS, nectin were obtained from Sigma-Aldrich. immunological synapse; ITSM, immunoreceptor tyrosine-based switch motif; KD, knockdown; MS, mass spectrometry; MTS, Aqueous One Solution Cell Proliferation; Cell culture, transfection, and stimulation NYU, New York University; PD-1, programmed cell death 1; PDL1, PD-1 ligand 1; SEE, Staphylococcus enterotoxin E; siRNA, small interfering RNA; TIL, tumor- Primary human T cells were isolated from whole blood using RosetteSep infiltrating lymphocyte; 39-UTR, 39-untranslated region; WT, wild type. (STEMCELL Technologies). Cells were maintained in enriched media at 5% CO2 at 37˚C. Primary murine T cells were isolated from spleens of Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 10- to 12-wk-old mice, followed by negative selection using Dynabeads www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800839 2 EFHD2 IS REQUIRED FOR T CELL CYTOTOXICITY (Invitrogen) T cell isolation kit. Jurkat T cells and Raji B cells were Flow cytometry obtained from the American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FBS and 100 U/ml peni- T cells were stained with fluorescently conjugated Abs specific for PD-1 in cillin and streptomycin. MC38 cells were provided by Benjamin Neel FACS Buffer, washed, and fixed in 1% paraformaldehyde. Events were (New York University [NYU]) and maintained in RPMI 1640 medium recorded using FACSCalibur (BD Biosciences) and analyzed using FlowJo supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. software. To obtain single-cell suspensions from tumors, tissues were HEK 293T cells were obtained from the American Type Culture Col- digested and cells were blocked with anti-FcR prior to staining with Abs against CD3, PD-1, CD44, CD8, CD4, and CD45 (BioLegend). For anti- lection and maintained in 5% CO2 at 37˚C in DMEM media supple- mented with 10% FBS and 100 U/ml penicillin and streptomycin. DNA Foxp3 (BioLegend) staining, cells were fixed and permeabilized using expression constructs were introduced into the cells by nucleofection True-Nuclear Transcription Factor Buffer Set (BioLegend). Samples were (Lonza Bioscience) with efficiency of 50–70%. Cells were stimulated collected on a BD LSR II Flow Cytometer (BD Biosciences), and data with magnetic beads (ratio of 1:3 cells per bead), which were conjugated were analyzed using FlowJo software. with the following protein combinations (the ratio in parentheses Static adhesion assay indicates the relative concentration of each protein): anti-CD3/IgG1 (1:3), anti-CD3/PDL1-Fc/IgG1 (1:2:1), anti-CD3/PDL2-Fc/IgG1 (1:2:1), Static T cell adhesion to fibronectin-coated plates was performed as anti-CD3/anti-CD28/IgG1 (1:1:2), or anti-CD3/anti-CD28/PDL1-Fc reported earlier (21). (1:1:2). Constructs were introduced into HEK 293T cells using Super- Fect Transfection Reagent (Qiagen) according to the manufacturer’s Conjugate formation assay protocol. SEE (Toxin Technology)-loaded Raji B cells (1.5 3 106) were mixed with 6 Abs Jurkat T cells (1.5 3 10 ), plated on poly-L-lysine (5 mg/ml)–coated glass bottom culture 35-mm plates (ibidi), and subjected to brief centrifugation Anti-CD3 (UCHT1), PDL1-Fc, and PDL2-Fc were purchased from R&D prior to imaging.